Normal oxidative metabolism leads to the generation of various redox forms of molecular[unreadable] oxygen, termed reactive oxygen species (ROS), that are generated over a range of concentrations[unreadable] within cells. Low levels of ROS production are important for normal signaling mechanisms, while[unreadable] higher levels of ROS production can lead to oxidant stress, a condition under which the flux of ROS[unreadable] exceeds antioxidant capacity. A key determinant of the response to oxidant stress is the cell's capacity[unreadable] to counter increased ROS generation by adaptively increasing the production of NADPH, the principal[unreadable] source of reducing equivalents for the reduction of oxidized glutathione. The primary cytosolic enzyme[unreadable] required for NADPH synthesis is glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting[unreadable] enzyme in the pentose phosphate pathway. We have previously demonstrated the importance of this[unreadable] enzyme in maintaining normal endothelial function, including nitric oxide (NO) bioactivity, in the face of[unreadable] oxidant stress. In this proposal, we hypothesize that G6PD and its enzymatic product NADPH are the[unreadable] key regulators of the thiol redox state of the endothelial cell, are essential for normal oxidant signaling[unreadable] and endothelial function, adaptively respond to increased ROS generation to maintain a state of[unreadable] compensated oxidant stress, and when oxidant stress exceeds this adaptive capacity are responsible[unreadable] for the conversion of compensated oxidant stress to uncompensated oxidant stress in the endothelial[unreadable] cell. To test this hypothesis, we will 1) assess the relationship between NADPH and the redox state of[unreadable] endothelial thiol pools and their enzymatic determinants; 2) evaluate the effect of NADPH on the[unreadable] synthesis, metabolism, and bioavailability of endothelial NO and its S-nitroso-derivatives; 3) determine[unreadable] the role of G6PD and NADPH in the adaptive response to oxidant stress in endothelial cells, including[unreadable] their role in supporting the state of compensated oxidant stress; and 4) evaluate the relationship[unreadable] between thiol redox state and endothelial NO bioactivity under conditions of normal oxidant signaling,[unreadable] compensated oxidant stress, and uncompensated oxidant stress in vivo. These studies should shed[unreadable] light on the critical role of G6PD and NADPH in regulating the thiol redox state of the endothelial cell[unreadable] and its ability to adapt to oxidant stress in an effort to maintain normal endothelial function.